Laboratory evolution of virus-like nucleocapsids from nonviral protein cages

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 115; no. 21; pp. 5432 - 5437
• Main Authors: Terasaka, Naohiro, Azuma, Yusuke, Hilvert, Donald
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 22-05-2018
• Subjects: Bacteria - enzymology; Bacteria - genetics; Bioengineering; Biological Sciences; Cages; Capsid Proteins - chemistry; Capsid Proteins - genetics; Capsid Proteins - metabolism; Cationic peptides; Deoxyribonucleic acid; Directed Molecular Evolution; DNA; Evolution; Genomes; Life cycles; Lumazine synthase; Models, Molecular; Molecules; mRNA; Multienzyme Complexes - chemistry; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; Nuclease; Nucleocapsid - chemistry; Nucleocapsid - genetics; Nucleocapsid - metabolism; Nucleocapsids; Optimization; Packaging; Peptide Fragments - metabolism; Peptides; Proteins; Ribonucleic acid; RNA; Virions; Virus Assembly; Viruses; nucleocapsid; synthetic biology; protein cage; directed evolution
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1800527115

Viruses are remarkable nanomachines that efficiently hijack cellular functions to replicate and self-assemble their components within a complex biological environment. As all steps of the viral life cycle depend on formation of a protective proteinaceous shell that packages the DNA or RNA genome, bottom-up construction of virus-like nucleocapsids from nonviral materials could provide valuable insights into virion assembly and evolution. Such constructs could also serve as safe alternatives to natural viruses for diverse nano- and biotechnological applications. Here we show that artificial virus-like nucleocapsids can be generated—rapidly and surprisingly easily—by engineering and laboratory evolution of a nonviral protein cage formed by Aquifex aeolicus lumazine synthase (AaLS) and its encoding mRNA. Cationic peptides were appended to the engineered capsid proteins to enable specific recognition of packaging signals on cognate mRNAs, and subsequent evolutionary optimization afforded nucleocapsids with expanded spherical structures that encapsulate their own full-length RNA genome in vivo and protect the cargo molecules from nucleases. These findings provide strong experimental support for the hypothesis that subcellular protein-bounded compartments may have facilitated the emergence of ancient viruses.